Analysis of virulence of Pseudomonas syringae on Arabidopsis thaliana

Pseudomonas syringae pv. maculicola ES4326 (PsmES4326) is a phytopathogen that causes leaf spot disease on the host plant Arabidopsis thaliana. My project was to identify PsmES4326 genes that encode secreted virulence factors that contribute to disease symptoms on Arabidopsis leaves. I isolated eight bacterial mutants that elicit attenuated disease symptoms by mutagenizing the bacterial chromosome with the transposon TnphoA and screening individual mutants on Arabidopsis leaves. Six of the mutants grew as well as PsmES4326 in Arabidopsis leaves while two of the mutants grew to significantly lower titers in leaves. Sequence analysis of the eight PsmES4326::TnphoA mutants revealed that five mutants had insertions in genes that were similar to previously known bacterial genes and three mutants had insertions in novel genes. To investigate whether plant defenses contribute to disease symptoms in infected plants, I measured the expression of the Arabidopsis pathogenesis-related (PR) genes PR-1, PR-5, and BGL2 and the induction of camalexin, an antimicrobial phytoalexin produced by Arabidopsis. I found that the expression patterns of the PR genes and camalexin in Arabidopsis leaves were similar in response to the mutant bacterial strains and wild type PsmES4326.;I describe the further characterization of a PsmES4326::TnphoA virulence mutant that has a transposon insertion in a gene that is similar to members of the degP (htrA) gene family. This mutant grows to significantly lower titers than Psm/ES4326 in Arabidopsis leaves. Complementation analysis of this mutant and null alleles of the PsmES4326 degP gene revealed that mutation of the degP gene is responsible for the attenuated virulence phenotypes observed in Arabidopsis. Additional phenotypes of the PsmES4326 degP mutants, including colony morphology and sensitivity to oxidants, are presented. The comparison of the virulence phenotypes of PsmES4326 degP mutants and degP mutants of several mammalian pathogens is discussed.